ABSTRACT Regulatory T (Treg) cells play a fundamental role in enforcing peripheral immunological tolerance to self-antigens, commensal flora, and innocuous foreign antigens. The transcription factor Foxp3 is indispensable to Treg cell differentiation and immunoregulatory functions. Loss of function mutations in Foxp3 precipitate a severe autoimmune inflammatory disorder, while acquired deficiency in chronic inflammatory and autoimmune conditions destabilizes Treg cells and sustains disease chronicity and severity. Foxp3-deficient Treg (?Treg) cells lack suppressor function and manifest an effector T (Teff) cell?like phenotype. Nevertheless, they continue to a core regulatory transcriptome, indicating the potential to restore their regulatory function. In agreement with this hypothesis, we have recently demonstrated that Foxp3 deficiency dysregulates metabolic checkpoint mTORC2 signaling and gives rise to augmented aerobic glycolysis and oxidative phosphorylation. Specific deletion of the mTORC2 adaptor gene Rictor in ?Treg cells greatly ameliorated disease in Foxp3-deficient mice, improved ?Treg cells suppressive capacities, and suppressed their Teff cell?like glycolytic and respiratory programs. These findings established for the first time the potential to reprogram ?Treg cells in favor of enhanced tolerance, an approach applicable to both monogenic and common immune dysregulatory diseases. More recently, we have established that the ?Treg cells are heterogenous, ranging along a spectrum from cells with high regulatory potential to ones more advanced towards a Teff cell-like phenotype. Based on these results, we postulate that Foxp3 deficiency destabilizes ?Treg cells towards Teff cell-like programs in a step-wise process that can be reversed by precision interventions to re-establish their regulatory functions. Specifically, the degeneration of ?Treg cells in to Teff-like cells involves the transition from a CD25+Foxp1high cell population into CD25?Foxp1low activated Teff-like cells that ultimately emerge as ex-Treg cells lacking in a Treg cell epigenetic imprint. The role of IL-2/CD25 and Foxp1 in this transition will be examined using functional and genetic approaches (Aim 1). Under Aim 2, we will examine the metabolic checkpoints involved in this transition and the capacity of targeted combinatorial metabolic and functional interventions to reprogram ?Treg cells in favor of tolerance. Under Aim 3, we will employ insights gained from our preliminary and proposed studies to reinforce the regulatory functions of Foxp3-sufficient Treg cells in the context of autoimmune and inflammatory disease models. The proposed analysis of ?Treg cell subsets and their modulation by single and combinatorial interventional strategies represents a novel approach to restoring immune tolerance in monogenic and common immune dysregulatory diseases, including autoimmune and allergic disorders and graft versus host disease.